CN102277411A

CN102277411A - Model for screening 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD) inhibitor and novel application of IspD inhibitor domiphen bromide

Info

Publication number: CN102277411A
Application number: CN2010101969226A
Authority: CN
Inventors: 肖春玲; 高鹏; 杨延辉; 熊小椒; 关艳; 甘茂罗; 郝雪秦; 刘忆霜
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2010-06-10
Filing date: 2010-06-10
Publication date: 2011-12-14
Anticipated expiration: 2030-06-10
Also published as: CN102277411B

Abstract

The invention relates to a reagent set for screening a 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD) inhibitor. The reagent set comprises methylerythritol phosphate (MEP), cytidine triphosphate (CTP), IspD, enzyme reaction buffer solution, an ion reagent and a reagent for determining pyrophosphoric acid (PPi). The invention also relates to a method for screening the IspD inhibitor. By the method, domiphen bromide with anti-tuberculosis activity is screened from more than 3,000 compounds. The invention also relates to application of the domiphen bromide to the in-vivo/in-vitro inhibition of Mycobacterium tuberculosis and the preparation of anti-tuberculosis medicines.

Description

The new purposes of lspD inhibitor screening model and IspD inhibitor oradol

Technical field

The present invention relates to chemicals and drug screening field, particularly, the present invention relates to a kind of screening model of tubercule bacillus enzyme IspD inhibitor, and IspD inhibitor oradol is used to prepare the purposes of antituberculosis therapy medicine.

Background technology

Tuberculosis (Tuberculosis is designated hereinafter simply as " TB ") is to infect the chronic infection disease that causes by mycobacterium tuberculosis (Mycobacterium tuberculosis), and human life's health in serious harm.Now the population in the whole world 1/3rd has infected mycobacterium tuberculosis, and annual newly-increased tuberculosis case 9,270,000 examples, and is dead 3,000,000, becomes and AIDS, malaria and the three big transmissible diseases that claim.China is one of the high burden of 22 tuberculosis in whole world country, has 5.5 hundred million populations to infect TB, and patient's number occupies the second place of the world up to 5,500,000, increases tuberculosis patient 4000 many cases every day newly.

Mycobacterium tuberculosis belongs to Mycobacterium, is pathogenic bacteria lungy.The resistance of mycobacterium tuberculosis has become one of the most serious problem that is faced in the current tuberculotherapy, the mycobacterium tuberculosis of single medicine resistance, multidrug resistance even serious resistance (extreme resistance) constantly occurs and propagates, and makes traditional anti-TB medicine lose original curative effect.In in the past three, 40 years, fail to develop real effectively novel anti TB medicine.And the protection ratio of bacille Calmette-Guerin vaccine also has been lower than 50% in developed country, and is then lower in China.Threaten in order to tackle tuberculosis, press for the new anti-TB medicine of development.

All the time, cell walls is the first-selected target spot of screening anti-bacterial drug.The cell walls of tubercule bacillus is most important for its growth and breeding, has numerous enzyme systems to participate in the synthetic and assembling process of each composition of cell walls, potential the novel targets of a plurality of antitubercular agents.And wherein most enzymes is that institute is non-existent in the human body, so the medicine of targeted cells wall has and selects toxicity preferably.

Result of study in recent years shows: have a 2-methyl-erythritol phosphoric acid (methylerythritol phosphate in some bacterium, protozoon and plant materials, MEP) approach is used for precursor substance isovaleryl tetra-sodium (the isopentenyl diphosphate of synthetic isoprenoid, IPP) or the dimethylallyl tetra-sodium (dimethylallyl diphosphate, DMAPP).This approach is different from mevalonic acid (MVA) approach in Mammals and the plant endochylema fully.MEP approach synthetic isoprenoid and derivative thereof are most important in biological metabolism and cell walls are synthetic, so the MEP approach has become the focus of medicine target research.A certain transgenation or disappearance with escherichia coli or intestines salmonella MEP approach can cause lethal mutation, confirm that the MEP approach is essential to the existence of bacterium.Because the MEP approach extensively exists in pathogenic agent, and be not present in the people, in the animal and plant endochylema, make that the enzyme of this approach of catalysis becomes potential, have optionally molecule target position simultaneously again.

The MEP approach comprises 8 enzymes altogether participates, and is respectively DXS, IspC, IspD, IspE, IspF, IspG, IspH, Idi.IspD (2-C-methyl-D-erythritol-4-phosphateCytidyltransferase wherein, 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase) is the key gene in this approach, is responsible for MEP and CTP (cytidine) catalyzed reaction are generated CDP-ME (as shown in Figure 1).Dean Crick has carried out temperature-sensitive mutation to this gene, proof is this transgenation tubercule bacillus (Hyungjin Eoh of can't surviving really, Amanda C.Brown, Lori Buetow, et al.Brennan, and Dean C.Crick.Characterization of the Mycobacterium tuberculosis4-Diphosphocytidyl-2-C-Methyl-d-Erythritol Synthase:Potential for Drug Development.J Bacteriol.2007,189 (24): 8922-8927).But the report that does not still have at present the IspD inhibitor might be found the antitubercular agent of brand-new mechanism of action by the IspD inhibitor of screening tubercule bacillus.

Oradol (Domiphen Bromide, DMB) cation type surfactivity wide-spectrum bactericide.It is applicable to assisting therapy and skin, the sterilization of instruments etc. of oral cavity, throat infection.Still there be not at present the report of oradol as antitubercular agent.

Summary of the invention

In order to screen the IspD inhibitor, the present invention is a target with IspD, utilizes its enzyme activity to set up the IspD inhibitor screening model, and estimates the tuberculosis activity of screening product, finds that finally oradol has tubercle bacillus resistant activity.Particularly,

One aspect of the present invention relates to a kind of composite reagent of the IspD of screening inhibitor, and it comprises MEP, CTP, IspD and treats SCREENED COMPOUND; Also comprise enzyme reaction buffer solution and ion reagent, wherein said ion reagent comprises MgCl ₂, NaF and DTT (dithiothreitol (DTT)); Comprise the reagent of the PPi that mensuration MEP, CTP and IspD reaction is generated in addition, described reagent is preferably beta-mercaptoethanol and ammonium molybdate.

Wherein said IspD extract to obtain from the biology that can produce IspD or utilizes molecular biology method to obtain according to the genome sequence of above-mentioned biology.

Preferably, IspD extracts acquisition or utilizes molecular biology method to obtain according to the mycobacterium tuberculosis genome sequence from mycobacterium tuberculosis.

In one embodiment of the invention, be masterplate with tubercule bacillus H37Rv genome, respectively with 5 '-GTTCATC CATATGGTCAGGGAAGCGGGCGAAGTAGTTGCG-3 ' (SEQ IDNO:1) and 5 '-GTCTTAT CTCGAGCCCGCGCACTATAGCTTGGGCCAGC-3 ' (SEQID NO:2) is a primer, obtain the ispD gene order by pcr amplification, the clone also transforms, obtain to efficiently express the proteic intestinal bacteria B121 of tubercule bacillus IspD (DE3), the IPTG inducible protein is expressed; get the expression product purifying, is used for screening.

Another aspect of the present invention relates to the method for screening IspD inhibitor.

The ultimate principle of this method is: synthetic (Fig. 1) of the synthetic precursor CDP-ME of IspD catalysis bacillus tubercle cell wall, in the reaction process, in the product C DP-ME that generates 1mol, discharge the tetra-sodium (PPi) of 1mol, the PPi growing amount directly reflects the situation that this enzymatic reaction is carried out, thereby can reflect the activity of IspD indirectly.

The mensuration of PPi growing amount can adopt radiometric method, and last single phosphoric acid of CTP is carried out radio-labeling, the radioactive intensity detection reaction process of the PPi that generates by reaction, but this method has radiocontamination, is unsuitable for extensive use; Also can adopt the inorganic phosphate enzyme that PPi is decomposed into single phosphoric acid, utilize color reaction to detect then, the shortcoming of this method is to have introduced other enzyme in this process, needs oppositely screening to get rid of false positive, has increased the screening step.

In the present invention, the mensuration of PPi preferably adopts absorbance method, reaction is according to being: under acidic conditions, pyrophosphate and ammonium molybdate form blue product when mercaptoethanol exists, the difference of the absorption value of this blue product and background (yellow) absorption value is linear with the product growing amount within the specific limits, growing amount (the Yu Kuang of can the be easy believable detection of this method PPi, NicolasSalem, Fangjing Wang, et al.A Colorimetric Assay Method toMeasure Acetyl-CoA Synthetase Activity:Application toWoodchuck Model of Hepatitis Virusinduced HepatocellularCarcinoma.J Biochem Biophys Methods.2007,70 (4): 649-655.).

This method may further comprise the steps:

A. with CTP, MEP, IspD, treat SCREENED COMPOUND, enzyme reaction buffer solution and ion reagent mix, the anabolic reaction system is hatched, and sets up positive control and negative control group simultaneously;

B. further add beta-mercaptoethanol and ammonium molybdate, hatch, the absorbance value of detection reaction system draws the IspD enzymic activity;

C. calculate according to absorbance value and treat SCREENED COMPOUND, and then obtain that the IspD activity is had inhibiting positive compound the active inhibiting rate of IspD.

Wherein the reaction final concentration of each compound is respectively MEP 100 μ M-1mM, CTP 100 μ M-1mM, and IspD 3.85pmol-385pmol treats SCREENED COMPOUND 1-100 μ g/ml; In one embodiment of the invention, the reaction final concentration of each compound is respectively MEP 250 μ M, CTP 500 μ M, and IspD 38.5pmol treats SCREENED COMPOUND 10 μ g/ml.

The composition of wherein said enzyme reaction buffer solution is the Tris-HCl of 50mM pH8.0, and the composition of wherein said ion reagent is the MgCl that is dissolved in the 10mM in the enzyme reaction buffer solution ₂, 20mM NaF and 1mM DTT.

Wherein beta-mercaptoethanol concentration is 1M, and its consumption is 0.1 reaction system volume; Wherein amine molybdate concentration is 2.5%, is dissolved in the H of 2.5M ₂SO ₄In, its consumption is 0.4 reaction system volume.

In one embodiment of the invention, the reaction system cumulative volume is 100 μ l, and wherein the final concentration of CTP and MEP is respectively 500 μ M and 250 μ M, ion reagent 10 μ l, and IspD38.5pmol treats SCREENED COMPOUND 10 μ g/ml.Above-mentioned system is hatched 40min at 37 ℃, and (2.5% ammonium molybdate is dissolved to 2.5M H to add 10 μ l color reagent A (1M beta-mercaptoethanol) and 40 μ l color reagent B ₂SO4) after, 37 ℃ are continued to hatch 10min, the absorbance value of microplate reader detection reaction system under the 590nm wavelength.

Wherein said positive controls is meant the IspD that adds inactivation in the reaction system, does not add and treats SCREENED COMPOUND; Wherein said negative control group is meant not add in the reaction system treats SCREENED COMPOUND.

The wherein said method of calculating inhibiting rate according to absorbance value is suc as formula shown in (I):

Inhibiting rate is considered as positive findings greater than 20%.

Utilize above screening method, the present invention screens from more than 3000 compounds and obtains 5 positive compounds, and positive rate is 0.17%, and wherein the oradol of 10 μ g/ml is 44% to the inhibiting rate of IspD.

Of the present invention also relate on the one hand again IspD inhibitor oradol in vivo/purposes of vitro inhibition mycobacterium; Preferably, described mycobacterium is a mycobacterium tuberculosis; In one embodiment of the invention, described mycobacterium tuberculosis is mycobacterium tuberculosis H ₃₇Rv.Because H ₃₇Rv is a representative strain lungy, therefore to H ₃₇The medicine that the inhibited medicine of Rv is regarded as having the tuberculotherapy prospect usually.

The invention still further relates to oradol and be used to prepare the purposes of antitubercular agent.

The invention still further relates to IspD and be used for screening the purposes of the composition of tuberculosis inhibitor in preparation.

The beneficial effect of the invention

The present invention utilizes the enzyme activity of IspD to set up the screening method of IspD inhibitor, and screening obtains 5 positive compounds from more than 3000 compounds, and wherein the oradol of 10 μ g/ml reaches 44% to the inhibiting rate of IspD.Experimental results show that oradol all has a tuberculosis activity with external in vivo, is new antitubercular agent likely.

Description of drawings

Fig. 1: MEP path synoptic diagram

Fig. 2: the catalytic reaction formula of IspD albumen

2-C-methyl D-erythritol-4-phosphoric acid (MEP) generates 4-cytidine diphosphate (CDP) 2-C-methyl D-erythritol (CDP-ME) by IspD catalysis under the condition that CTP exists, discharge PPi simultaneously.

Fig. 3: ispD gene clone checking electrophoretogram

Expression plasmid pET-28a/ispD has obtained the purpose fragment ispD of 720bp through Nde I and Xho I double digestion.Swimming lane 1:DNA marker; Swimming lane 2: the purpose fragment ispD of the 720bp that expression plasmid obtains behind double digestion

Fig. 4: ispD gene sequencing result

Fig. 5: SDS-PAGE and western blot checking result behind the IspD protein purification

Target protein IspD size is about 26KD.Left side figure is SDS-PAGE figure, and right figure is westernblot figure.Swimming lane 1: protein quantification Marker is respectively 80kd, 60kd, 40kd, 30kd, 20kd, 12kd from top to bottom; The protein I spD of swimming lane 2 and swimming lane 3:16 ℃ purifying; Swimming lane 4: protein quantification Marker is respectively 94kd, 62kd, 47kd, 30kd, 24kd, 16kd from top to bottom; Swimming lane 5 and swimming lane 6: the western of purifying protein IspD detects.

Fig. 6: tetra-sodium (ppi) concentration standard curve

Embodiment

Below in conjunction with embodiment embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used to illustrate the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person among the embodiment carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.

The clone of embodiment 1:ispD

With tubercule bacillus H ₃₇Rv (professor Zhang Jianyuan of institute of tuberculosis is so kind as to give) genome is a template, respectively with F1:5 '-GTTCATC CATATGGTCAGGGAAGCGGGCGAAGTAGTTGCG-3 ' (SEQID NO:1) and R1:5 '-GTCTTAT CTCGAGCCCGCGCACTATAGCTTGGGCCAGC-3 ' (SEQ ID NO:2) is a primer, by pcr amplification ispD gene order; Adopt the amplification of DNA agarose gel electrophoresis testing goal gene, reclaim the purpose fragment ispD (Fig. 3) of 720bp size then.

The PCR reaction system:

The pcr amplification condition: 95 ℃ of pre-sex change 5min, then with 95 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ are extended 2min, carry out 32 reaction cycle, and last 72 ℃ are extended 10min.

The goal gene ispD that obtains is connected with plasmid pET28a, connects the competent cell of product transformed into escherichia coli DH5 α.Obtain positive colony by blue hickie screening, positive colony is carried out PCR and the evaluation of plasmid double digestion, and extraction PCR detects and double digestion detects the plasmid (pET-28a/ispD) that is all positive strain, carry out sequencing, the result is shown in SEQ ID NO:3 (Fig. 4), and wherein the 47th to the 744th is the ispD sequence.

Extract the plasmid pET-28a/ispD in the positive strain, transformed into escherichia coli BL21 (DE3) plysS bacterial strain, structure efficiently expresses BL21 (DE3) plysS of tubercule bacillus IspD, carry out bacterial classification through the engineering bacteria that PCR detects and the double digestion detection is all positive and sequencing result is correct with 20% glycerine and preserve, frozen in-80 ℃.

The proteic expression of embodiment 2:IspD

With the proteic intestinal bacteria B121 of efficiently expressed tubercule bacillus IspD (DE3) streak inoculation of frozen embodiment 1 preparation in the LB flat board that contains 100 μ g/ml kantlex (Kan) and 37 μ g/ml paraxin (Ch1), 37 ℃, incubated overnight, picking list colony inoculation is in the LB liquid nutrient medium that contains 100 μ g/ml Kan and 37 μ g/ml Ch1,200r.p.m., 37 ℃, incubated overnight; Overnight culture was inoculated in the fresh LB liquid nutrient medium that contains 100 μ g/ml Kan and 37 μ g/mlCh1 by 1: 50, and 200r.p.m., is cultured to thalline OD by 37 ℃ ₆₀₀≈ 0.5; Add in the culture IPTG to final concentration be 1mmol/L, add 50% yeast extract to final concentration 0.5%.16 ℃, 200r.p.m. cultivates 12h, induces the proteic expression of IspD.

Proteic purifying of embodiment 3:IsDD and detection

Condition cultivation by embodiment 2 can efficiently express the proteic e. coli bl21 of tubercule bacillus IspD (DE3) plysS, and abduction delivering IspD albumen; The centrifugal 10min of 10000 * g collects thalline; Lysis buffer suspended bacteria somatocyte, ice bath, 400W, 3s/8s, 99 ultrasonication somatic cells, 4 ℃ of centrifugal 1h of following 14000 * g collect supernatant liquor; Use

The prime system, under the condition of pH8.0, (GE company 17-5247-01), carries out gradient elution with damping fluid to Hi-trap affinity column on the supernatant liquor, collects elutriant; The sample collected adds 15ml ultra-filtration centrifuge tube (10kDa, MilliPore company), 4 ℃ of centrifugal 15min of following 5000g, to sample size less than 2.5ml; Sample desalination after using PD-10 desalting column and desalination damping fluid with ultrafiltration.-80 ℃ of preservations.

By the protein I spD (as shown in Figure 5) behind SDS-PAGE and the Western Blot electrophoresis detection purifying.

Embodiment 4: enzyme activity assay

Reaction system:

The 10 μ l ion reagent (MgCl that contain final concentration 10mM ₂, 20mM NaF and 1mM DTT); The IspD of the purifying that the embodiment 3 of 1 μ g obtains; CTP and MEP (its final concentration is respectively 500 μ M and 250 μ M).

Enzyme reaction buffer solution is 50mM Tris-HCl (pH8.0), and the reaction system cumulative volume is 100 μ l.

If the IspD that does not add IspD or add heat inactivation in contrast.

Establish simultaneously and add 8 different concns (0 μ M, 25 μ M, 50 μ M, 75 μ M, 100 μ M, 150 μ M, 200 μ M, 250 μ M) 100 μ l PPi draw the typical curve of absorption value and PPi concentration in determination of activity, simulate the formula (referring to Fig. 6) of relation line sigmoid curve by Excel, thereby calculate the reaction process (as Fig. 6) of enzyme according to formula.

Determination of activity:

Above-mentioned system is hatched 40min at 37 ℃, and (2.5% ammonium molybdate is dissolved to 2.5M H to add 10 μ l color reagent A (1M beta-mercaptoethanol) and 40 μ l color reagent B ₂SO4) after, 37 ℃ are continued to hatch 10min, the absorbance value of microplate reader detection reaction system under the 590nm wavelength.

The result:

With the absorption value reference standard curve of sample, calculate the concentration of the PPi of reaction generation.The concentration of the PPi that the concentration of the PPi that generates may be generated divided by complete reaction is as reaction process.The substitution formula:

Concentration * 100% of the PPi that the concentration of the PPi of reaction process=generation/complete reaction generates

When reaction process greater than 40%, it is normal that the activity of enzyme promptly is considered to.

The screening of embodiment 5:IspD inhibitor

Principle, reaction system and the activity determination method of screening method therefor are with embodiment 4, and concrete screening grouping sees Table 1.

Table 1

Annotate: be the reaction final concentration in the bracket.

Compound (comprising 2458 of 734 of natural products and chemosynthesis compounds) to different sources in above-mentioned screening system screens, and the method for calculation of enzyme inhibition rate are as follows:

In this screening system come out owing to still there is the IspD inhibitor, with the IspD of inactivation as positive controls, the absorption value minimum that it provides, enzyme inhibition rate be a maximum; With the fully normal negative contrast of enzymatic reaction system, the absorption value maximum that this group provides, inhibiting rate minimum.By calculating the enzyme inhibition rate of waiting to sieve sample, when inhibiting rate promptly is considered to positive findings greater than 20%.

The selection result:

Obtain 5 positive compounds from middle screening of 3192 compounds (comprise 2458 of 734 of natural products and chemosynthesis compounds, wherein 2050 be that this institute is synthetic, and 258 are provided by China Medicine University, 150 provided by Zhongshan University), positive rate is 0.17%.Wherein the oradol of 10 μ g/ml is 44% to the inhibiting rate of IspD.

The evaluation of 6 screening models of embodiment

Be widely used in the quantitative technique parameter of estimating the screening model quality at present and have four: the variation coefficient (CV) and Z '-factor at the bottom of signal/background ratio (S/B), signal/noise ratio (S/N), the code book, each parameter calculation formula is as follows:

Each parameter value of this screening model sees Table 2.

Table 2

As can be seen from Table 2, this screening method background is low, noise is little, good reproducibility, reliable results.

Embodiment 7: the Tuberculosis in vitro nuclear activity of oradol is measured

The tuberculosis activity determination method is: adopt aseptic 48 orifice plates to carry out the tuberculosis determination of activity of oradol.Each hole adds the medicine with 2 times of concentration substratum (Middlebrook, BD Difco) dilution respectively.Each positive compound that embodiment 5 screenings obtain is made the first solution of proper concn, be diluted to two times of concentration of each compound used therefor with substratum (2 *), every kind of each 10 gradient of positive compound, the every hole of 48 orifice plates adds 100 μ l, and each positive compound final concentration is: 50.0,40.0,20.0,10.0,5.0,2.5,1.25,0.625,0.315 and 0.156 μ g/ml.Contrast drug isoniazid (INH, Sigma company) final concentration is: 40.0,20.0,10.0,5.0,2.5,1.25,0.625,0.312,0.156,0.078,0.039 and 0.019 μ g/ml.Then add mycobacterium tuberculosis H37Rv, 100 μ l are inoculated in every hole, and every pore fungi amount is 4 * 10 ^-3Mg.Every plate is all established 2 growth positive control hole and two growth negative control holes with the alternative substratum of distilled water of not containing antimicrobial drug, and 48 orifice plates are added a cover the back on every side with the scotch tape sealing, places wet box to hatch for 37 ℃.Observe positive growth control hole and negative growth control hole after the 3rd day.When observing both clear and definite difference being arranged, the quantity and the form of each test holes bacterial growth are observed, judge that inhibition or resistance also write down the result, after the 7th day again observed and recorded once confirm.

Measurement result sees Table 3.

Table 3

Conclusion: oradol is to tubercule bacillus H ₃₇The minimum inhibition concentration of Rv (MIC) is 5.0-10 μ g/ml; The positive control vazadrine is to tubercule bacillus H ₃₇The MIC of Rv is 0.1 μ g/ml;

Embodiment 8: tuberculosis activity rating in the body

The tuberculosis activity that adopts chmice acute aerosol infection tuberculosis model evaluation embodiment 5 to screen the positive compound that obtains.Method is standard operating procedure (the Lisa A.Collins according to aerosol infection device (099C A4224Inhalation Exposure System), ScottG.Franzblau.Microplate Alamar Blue Assay versus BACTEC 460System for High-Throughput Screening of Compounds againstMycobacterium tuberculosis and Mycobacterium avium.Antimicrobial Agents And Chemotherapy.1997,41 (5): 1004-5) infecting mouse, infective dose 50-100CFU/ are only.Infect back 3 days, dead 3 mouse in 10 natural gift other places, do the lung tissue live bacterial count.Infect and began to give pharmacological agent on the 10th day, be subjected to reagent thing DMB4mg/kg, positive control drug Rifampin 10mg/kg, vazadrine 25mg/kg, establishing CMC does not simultaneously have the medicine control group, and 6 every group, all oral administration gavage administrations, Mon-Fri weekly is administered once every day, and totally 5 times weekly, totally 15 dosage.Infect back execution in the 30th day and respectively organize mouse, weigh, aseptic technique is dissected down, does the lung tissue live bacterial count.

Measurement result sees Table 4.

Table 4

Conclusion: under the dosage of 4mg/kg, the DMB experimental group has shown the intravital tubercule bacillus titre of mouse and has descended, through two sample t checks, the DMB group is compared with control group, there is the significant difference on the statistics in p＜0.001, and promptly DMB has shown the tuberculosis activity.

Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that according to disclosed all instructions can carry out various modifications and replacement to which details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.

Sequence table

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＜120〉the new purposes of IspD inhibitor screening model and IspD inhibitor oradol

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Claims

1. be used for the reagent set of 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase inhibitor screening, comprise 2-methyl-erythritol phosphoric acid, cytidine and 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase.

2. reagent set according to claim 1 also comprises enzyme reaction buffer solution and ion reagent, and wherein said ion reagent comprises MgCl ₂, NaF and DTT.

3. reagent set according to claim 1, also comprise the reagent of measuring 2-methyl-erythritol phosphoric acid, cytidine and 2-C-methyl D-erythritol-tetra-sodium that the reaction of 4-isopentenyl monophosphate pyrimidine transferase is generated, described reagent is preferably beta-mercaptoethanol and ammonium molybdate.

4. reagent set according to claim 1, wherein said 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase be from the biology that can produce 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase, extract to obtain or utilize molecular biology method to obtain according to the genome sequence of aforementioned biology.

5. use the method for the described reagent set screening of claim 1-4 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase inhibitor, may further comprise the steps:

A. with cytidine, 2-methyl-erythritol phosphoric acid, 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase, treat SCREENED COMPOUND, enzyme reaction buffer solution and ion reagent mix, the anabolic reaction system, hatch, set up positive control and negative control group simultaneously;

B. measure the amount of the tetra-sodium that is generated in a reaction, draw 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase activity; Be preferably adding beta-mercaptoethanol and ammonium molybdate, hatch, the absorbance value of detection reaction system draws 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase activity;

C. calculate according to absorbance value and treat SCREENED COMPOUND, and then obtain that 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase activity is had inhibiting positive compound 2-C-methyl D-erythritol-active inhibiting rate of 4-isopentenyl monophosphate pyrimidine transferase.

6. method according to claim 5, wherein the reaction final concentration of each compound is respectively 2-methyl-erythritol phosphatase 11 00 μ M-1mM, cytidine 100 μ M-1mM, 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase 3.85-385pmol is treated SCREENED COMPOUND 1-100 μ g/ml; Preferred concentration is 2-methyl-erythritol phosphoric acid 250 μ M, cytidine 500 μ M, and 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase 38.5pmol is treated SCREENED COMPOUND 10 μ g/ml.

7. method according to claim 5, wherein said positive controls are meant the 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase that adds inactivation in the reaction system, do not add and treat SCREENED COMPOUND; Wherein said negative control group is meant not add in the reaction system treats SCREENED COMPOUND.

8. method according to claim 5, the wherein said method of calculating inhibiting rate according to absorbance value is suc as formula shown in (I):

Oradol in vivo/purposes of vitro inhibition mycobacterium, described mycobacterium is preferably mycobacterium tuberculosis, most preferably is mycobacterium tuberculosis H ₃₇Rv.

10. oradol is used to prepare the purposes of antitubercular agent.

11.2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase is used for screening the purposes of the composition of tuberculosis inhibitor in preparation.